The present invention relates to a method for propagating fungi using solid state fermentation (SSF). SSF is particularly suitable for propagating fungi which are used for food, medicine, or health purposes. The kinds of fungi which can be propagated by SSF include, but not limited to, Cordyceps sinensis, Trametes versicolor, Antrodia camphorata, Agaricus Blazei, and Ganoderma Lucidum. The present invention also relates to the formulations and preparations of SSF media.
Cordyceps sinensis is a parasitic fungus that has been used as a traditional Chinese medicine since ancient times. It is particularly famous for treating patients with kidney failure and asthma. It is also known for its anti-tumor effects.
Recently, Lin et al., J. Lab. Clin. Med., 133:55-63 (1999), reported a finding of an active compound named xe2x80x9cH1Axe2x80x9d in the fruiting body of Cordyceps sinensis. H1A is a derivative of ergosterol having the chemical formula of: 
Ergosterol is (3xcex2, 22E)-Ergosta-5,7,22-trien-3-ol. H1A appears to have pharmacological effects on the immune system, renal function, and cardiovascular system. It has also been known to have clinical effects on suppressing the activated human mesangial cells (HMC) and alleviating IgA nephropathy (Berger""s Disease), thus, preventing the disease from progressing to the uremia stage. Lin et al.""s J. Lab. Clin. Med., 133:55-63 (1999) article is herein incorporated by reference.
The authors of the J. Lab. Clin. Med., 133:55-63 (1999) article, Lin et al., have a U.S. patent (U.S. Pat. No. 5,582,828) disclosing a method for identifying and isolating H1A from Cordyceps sinensis, which is also herein incorporated by reference.
Wild Cordyceps sinensis is extremely rare and difficult to obtain. The conventional cultivation methods for Cordyceps sinensis include solid media stationary incubation, liquid media rotating shaking incubation, liquid-state fermentation, and submerged liquid-state fermentation. Solid media stationary incubation uses solid media, such as malt extract agar (MEA), potato dextrose agar (PDA), or YEA (yeast extract agar), as culture media. Liquid media rotating shaking incubation and liquid-state fermentation use liquid media, such as malt extract broth (MEB), potato dextrose broth (PDB), or YEB (yeast extract broth) as culture media. They are known to be expensive and produce low yields of microorganisms. Moreover, it is not known that Cordyceps sinensis produced by these methods can generate a sufficient amount of H1A.
Submerged liquid-state fermentation is known for its capability of generating a high yield of microorganisms. However, this method requires high capital investment due to high energy and waste output. Also, because the fermentation cycle of the submerged liquid-state fermentation is generally short, usually five to seven days, it is doubtful that the metabolites generated by this method are sufficient enough to be of significance for medicinal uses.
Solid state fermentation (SSF) has been known for the production of enzymes and organic acids used in the paper pulp bleaching treatment industry and for producing organic pesticides. It is also widely used for producing soy sauce, alcoholic beverages from fruit or unpolished rice, and Chinese sour cabbage. SSF refers to the growth of microorganisms on moist solid substrates in systems with a continuous gas flow without the presence of free liquid between substrate particles. SSF has never been used for fungal propagation, particularly for fungi belonging to the classes of Ascomycotina and Basidomycotina in Eumycota.
There are two common types of solid bases used in an SSF culture. The first type uses xe2x80x9cnatural solid materialsxe2x80x9d as a solid base. For example, food stuffs or agricultural byproducts are used as a solid base not only to provide a physical matrix for microbial growth but also to be used as the main source of microbial nutrients. The second type uses an xe2x80x9cinert solid support supplemented with nutrientsxe2x80x9d as a solid base. This type of solid base acts only as a physical support for microbial growth.
An SSF culture contains three phases, which are: (a) a solid phase which comprises the solid base as described above; (b) a liquid phase which is bound to the solid phase where intraparticle mass transfer occurs; and (c) a continuous gas phase. The gas phase controls the temperature and moisture of the SSF culture. It also provides oxygen for fungal growth.
In contrast to other fermentation methods, SSF requires low capital investment due to low energy and waste output. The base materials and media used in SSF are generally cheaper and simpler than other methods. In addition, the SSF medium generally contains low water content which not only reduces the risk of contamination but also offers a favorable condition for fungal growth, because it resembles the natural habitats for fungi. In fact, the SSF culture allows the fungal spores to proliferate because they can lay onto the surface of the solid base.
SSF is also known to have the following disadvantages. First, it is very difficult to monitor and control the parameters (such as moisture, pH, temperature, substrate concentration etc.) of the fermentation process in an SSF culture. Second, thus far the underlying scientific and engineering basis of SSF is still poorly understood. Finally, direct quantitative measurements for biomass in an SSF culture are difficult. In fact, many of the studies done so far on SSF have been done in either qualitative or empirical levels.
In the invention to be presented in the following sections, a method for propagating fungi using SSF will be described. The present invention not only will teach a useful SSF medium to grow fungi with high yields of metabolites but also will teach ways to monitor and control the parameters of the fermentation such as moisture, pH, temperature, and substrate concentration to maximize the production of fungi. The present invention also will provide direct quantitative and qualitative measurements of the biomass and active ingredients. Finally, the present invention will provide a method for effective production of Cordyceps Sinensis and its active substance H1A.
The present invention provides a method for propagating fungi, especially the kinds of fungi which can be used for food, medicine, and health purposes. The method applies solid state fermentation (SSF) to propagate fungi. The preferred kinds of fungi that can be used in SSF include, but are not limited to, Cordyceps sinensis, Trametes versicolor, Antrodia camphorata, Agaricus blazei, and Ganoderma lucidum, which are all within the classes of Ascomycotina and Basidomycotina. Among these fungi, Cordyceps sinensis is the most preferred kind.
It is preferred that before SSF is to be applied to the propagation of a fungus, certain pre-cultivation steps are to be followed. First, wild, healthy fungus isolates of proper quality are selected. Alternatively, fungus isolates stored in liquid nitrogen are activated. These fungus isolates are disinfected. Their mycelia are cut into pieces under sterile condition and placed into a solid culture medium in a glass tube or plate. The solid culture medium is made of appropriate culture media such as potato dextrose agar (PDA), yeast extract agar (YEA), malt extract agar (MEA), yeast malt agar (YMA), and peptone yeast glucose agar (PYG).
After the mycelia have multiplied to cover most of the medium, approximately 0.5 cm in diameter of the mycelia are cut and transferred toga flask containing a liquid culture medium. The liquid culture medium is made of appropriate culture media such as potato dextrose broth (PDB), yeast extract broth (YEB), malt extract broth (MEB), yeast malt broth (YMB), and peptone yeast glucose broth (PYGB). The mycelia are grown under rotating shaking conditions for about 8 days, preferably about 5-6 days. Then, the mycelia are transferred to a larger shaker flask containing the same or different liquid culture medium and incubated under reciprocating shaking conditions for no more than 6 days, preferably about 3-4 days. This is followed by aeration/agitation and further incubation for 4-5 days. At this stage, the cultivated mycelia are ready for the SSF.
The SSF medium suitable for fungi propagation comprises a carbon source, a nitrogen source, vitamins, and inorganic substances. Additionally, trace elements and organic substances can be added. The carbon source is derived from at least one of the following: starch, glucose, monosaccharide, polysaccharide, dextrin, maltose, saccharose, methyl cellulose, fructose, turanose, and corn powder. The nitrogen source is derived from at least one of the following: defatted soybean powder, peptone, yeast paste, yeast syrup, peanut cake powder, yeast powder, wheat bran, casein, calcium caseinate, and defatted beancake powder. Vitamins include, but are not limited to, vitamin B1, vitamin B6, and nicotinic acid. Inorganic substances include, but are not limited to, calcium sulfate and calcium carbonate. The preferred ratio of the carbon source (C) and the nitrogen source (N) is about 5:1 to 25:1 by weight.
During the SSF, pH, water content in the SSF medium, temperature, relative humidity, and light cycle are properly controlled. The pH is preferred to be controlled at pH 4.5 to 7. The temperature is preferred to be controlled at 22xc2x150xc2x0 C. The water content in the SSF medium is preferred to be between 40 and 70%. The relative humidity is preferred to be between 60 and 80%. The light cycle is preferred to be at 30% light and 70% dark.
The incubation period for fungus in the SSF culture is normally between 20 and 60 days, preferably between 30 and 50 days. The longer the incubation period, the greater the production of the mycelium dry weight. However, prolonged incubation of fungus in the SSF culture does not guarantee that the production of active material/metabolite is also proportionally increased. For instance, as shown in FIG. 2a (infra), when Cordyceps sinensis is incubated in the SSF culture, the mycelial dried weight (g) increases during the first 34 days of incubation and plateaued between 34 and 50 days. On the contrary, as shown in FIG. 2b (infra), the amount of H1A, which is a derivative of ergosterol, is peaked at 38 days after incubation in the SSF culture. The amount of H1A decreases significantly during prolonged SSF incubation.
Due to this discrepancy, high performance liquid chromatography (HPLC) analysis of the total nucleoside amount and the amount of H1A/ergosterol is used in addition to the measurements of dried mass to determine when would be the proper time to harvest the fungi. The total nucleoside content not only reflects the active life cycles of the fungus (i.e., the higher the nucleoside content, the greater the replication of the fungus) but also may relate to pharmacological activities (i.e., the amount of adenosine in Cordyceps sinensis has been postulated by Dr. Ming-Shi Shiao of the Veteran Hospital in Taipei, Taiwan, to be related to its pharmacological activities). For purposes of producing higher quantity of active metabolites, such as H1A/ergosterol from Cordyceps sinensis, the amount of H1A/ergosterol is monitored and analyzed by HPLC.
Alternatively, the mycelia of the fungus grown in SSF for about 6 to 14 days can be transferred to another SSF for continuous cultivation of the fungus. In other words, the propagation of fungus in SSF can be continued for many generations as long as fresh SSF is provided.
As a specific example, the present invention also provides a method for propagating Cordyceps sinensis in an SSF culture using an SSF medium. As described above, the SSF culture is preferred to be preceded by pre-cultivation of the mycelia first in a slant culture medium comprising a solid medium such as PDA, YEA, MEA, YMA, and PGY followed by incubation of the mycelia in a liquid culture containing a liquid culture media such as PDB, YEB, MEB, YMB and PYGB.
The SSF medium contains malt extract, yeast extract, peptone, glucose, water, a solid base, and calcium carbonate/gypsum. Examples of a solid base include, but not limited to, rice, coarse rice/unpolished rice, corn, wheat, nude wheat, oat, and oatmeal. It is preferable that the SSF medium contains 0.3-4% by weight of malt extract, 0.3-4% by weight of yeast extract, 0.1-2% by weight of peptone, 1-5% by weight of glucose, 30-70% by weight of water, 40-60% by weight of solid base, and 2% by weight of calcium carbonate or gypsum.
The SSF medium is made by first mixing malt extract, yeast extract, peptone, glucose, water, and a solid base together and heating them to boiling to form a solid base mixture. Then, the solid base mixture is allowed to cool down. Finally, calcium carbonate or gypsum is added to the cooled solid base mixture and the final mixture is granulated. The SSF granules are placed into a SSF bottle and sterilized. After the mycelia have been inoculated, aerated, and thoroughly mixed with the SSF granules, the SSF granules-containing bottle is sat on the shelf in the incubator until harvest time.
The SSF incubation period for Cordyceps sinensis is determined either by the total nucleoside content or the amount of H1A and/or ergosterol in the dried mycelium of Cordyceps sinensis, both monitored and quantified by BPLC. The mycelia of Cordyceps sinensis in SSF can be transferred to a fresh SSF so that the propagation of Cordyceps sinensis can be continued.
The present invention further provides two SSF media. The first SSF comprises a carbon source, a nitrogen source, vitamin, and inorganic substance. The carbon source comprises at least one of the following: starch, glucose, monosaccharide, polysaccharide, dextrin, maltose, saccharose, methyl cellulose, fructose, turanose, and corn powder. The nitrogen source comprises at least one of the following: defatted soybean powder, peptone, yeast paste, yeast syrup, peanut cake powder, yeast powder, wheat bran, casein, calcium caseinate, and defatted beancake powder. The vitamin comprises at least one of the following: vitamin B1, vitamin B6, and nicotinic acid. The inorganic substance comprises at least one of the following: calcium sulfate and calcium carbonate. The preferred carbon source (C) and nitrogen source (N) ratio is 5:1 to 25:1 by weight.
The second SSF medium comprises 0.3-4% by weight of malt extract (preferably 0.5-3%, and most favorably 2%), 0.3-4% by weight of yeast extract (preferably 0.5-3%, and most favorably 2%), 0.1-2% by weight of peptone (preferably 0.3-1%, and most favorably 0.5%), 1-5% by weight of glucose (preferably 2-4%, and most favorably 2%), 30-70% by weight of water (preferably 40-60%, and most favorably 50%), 30-70% by weight of solid base (preferably 40-60%, and most favorably 50%), and 0.3-4% by weight of calcium carbonate or gypsum (preferably 0.5-3%, and most favorably 2%).